The goal of my Ruth L. Kirschstein Fellowship application is to develop a tissue-engineered skeletal muscle construct for repair of cranial facia defects. We propose to investigate the mechanism by which the Connexin43 (Cx43) carboxyl terminal (CT) mimetic peptide CT1 modulates the wound healing response and promotes regenerative repair of injured muscle by a tissue-engineered construct. Cranial facial muscle loss due to disease or trauma can impair the ability to chew food or smile. Current surgical repair involves rearrangement of skeletal muscle, though these approaches often fail when the muscle atrophies or results in fibrosis. Attempts at regenerating skeletal muscle lead to some degree of new muscle formation, though fibrosis is a persistent problem. In skin, promiscuous TGF signaling leads to increased collagen deposition and scarring. In skeletal muscle, TGF is a potent inhibitor of differentiation. Cx43 has been shown to activate TGF signaling by competing with smad2 for -tubulin binding. Our joint group has shown that CT1 reduces inflammation and scarring in skin wounds, and reduces myofibroblast differentiation and preserves underlying muscle in rodent and porcine models of implant contraction capsules - all effects which appear to be antagonistic of TGF signaling. In preliminary data we have determined that CT1 reduces collagen deposition in a model of rat skeletal muscle repair by a 3D tissue-engineered construct containing autologous satellite cells (SCs). This application tests the hypothesis that targeting Cx43 function via CT1 or gene knockdown promotes skeletal muscle differentiation and reduces fibrosis in tissue engineered skeletal muscle constructs through reduced downstream TGF signaling. This hypothesis will be tested in three aims: In the first aim, we will conduct a cell culture study on mechanism to test the prediction that CT1 or Cx43 shRNA reduces proliferation and promotes differentiation of the C2C12 myogenic cell line in vitro via reduced downstream TGF signaling. In the second aim, we will conduct a study on mechanism in 2D monolayers and in an existing 3D collagen tissue-engineered construct using primary rat satellite cells (rSCs) to test the prediction that CT1 or Cx43 shRNA reduces downstream TGF signaling and results in reduced proliferation and increased differentiation in vitro. In the third aim, we will conduct an animal study to test the prediction that targeting Cx43 with CT1 or Cx43 shRNA in the 3D tissue-engineered construct containing rSCs promotes regenerative integration of injured muscle in a cranial facial defect model in vivo. This work will be carried out under NIDCR funded RO-1 project DE019355-01A2 led jointly by my research mentor Dr. Robert Gourdie, and a member of my dissertation committee Dr. Michael Yost. The other members of my committee include Drs. Martine LaBerge (Chair, Bioengineering Department, Clemson University) and Richard Swaja (Director, Clemson-MUSC Bioengineering Program). This work will contribute to the clinical need of providing an effective means of regenerating structure and function following significant injury to cranial facial skeletal muscle.